Tunable nitric oxide-releasing macromolecules having multiple nitric oxide donor structures

ABSTRACT

Provided here are nitric oxide-releasing compounds that include at least two different NO donor functional groups of the same class. In some embodiments, such nitric oxide-releasing compounds are macromolecules such as dendrimer and co-condensed silica. Pharmaceutical compositions, wound dressings, kits and methods of treatments are also provided herein.

RELATED APPLICATION DATA

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/526,918, filed Aug. 24, 2011, the disclosure ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compounds that release nitric oxide.More particularly, the present invention relates to macromolecularcompositions for tuning nitric oxide-release kinetics for therapeuticpurposes.

BACKGROUND OF THE INVENTION

It is known that nitric oxide possesses a broad-spectrum ofantimicrobial activity and may be used as an alternative to conventionalantibiotics for drug resistant bacteria. Furthermore, some recentstudies have demonstrated that nitric oxide may also play an importantrole in the wound healing process by promoting angiogenesis throughstimulation of vascular endothelial growth factor (VEGF) and increasedfibroblast collagen synthesis. See Schaffer M R, et al.,Diabetes-impaired healing and reduced wound nitric oxide synthesis: Apossible pathophysiologic correlation. Surgery 1997; 121(5):513-9; andShi H P, et al., The role of iNOS in wound healing. Surgery 2001; 130(2):225-9. Thus, nitric oxide presents a promising addition and/oralternative to the conventional antibiotic treatment for wound care.

Nitric oxide is a gas at ambient temperature and atmospheric pressure,and it has a short half-life in a physiological milieu. Several smallmolecule nitric oxide donor prodrugs have been developed which havecontributed greatly to the understanding of nitric oxide in a number ofdisease states. However, due to issues with stability, indiscriminateNO-release, monotypical nitric oxide release kinetics, and inability totarget specific tissue types, no clinically viable solutions currentlyexist for administering nitric oxide outside of its gaseous form.Reproducibly delivering the appropriate levels of nitric oxide for agiven therapeutic indication is important because release of largeamounts of nitric oxide may be toxic or create undesirable side effectssuch as decreases in angiogenesis or increased inflammation. Therefore,it has been challenging to use nitric oxide in a therapeutic setting,other than via exogenous application, particularly in topicalapplications wherein nitric oxide has concentration dependent effectsand benefits from delivery in a controlled and targeted manner.

Dendrimers are a family of hyperbranched macromolecules with multivalentsurfaces that enable the design of targeted therapeutics agent deliveryvehicles. For example, polyamidoamines, polyamines, polypeptides,polyesters and polyethers dendrimers have been utilized for a range ofbiomedical applications, including drug and gene delivery, biologicalimaging, and tissue engineering. Dendimers have been also been used asmacromolecular nitric oxide donors.

Inorganic-organic hybrid silica nanoparticles have also been exploredfor applications spanning separation, biological labelling, diagnostics,and carrier systems for the controlled delivery of drugs. The drugdelivery potential of silica particles has received much attentionbecause of their physical and chemical versatility and non-toxic nature.Other materials, including functionalized metallic nanoparticles, havealso been used in drug delivery. Such nanoparticles have also been usedas macromolecular nitric oxide donors.

SUMMARY OF THE INVENTION

Provided according to embodiments of the invention are nitricoxide-releasing macromolecules that include at least two different NOdonor structures of the same class. Also provided are pharmaceuticalcompositions that include a nitric oxide-releasing macromoleculeaccording to an embodiment of the invention, and methods ofadministering such macromolecules and/or compositions to a subject.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects, features and advantages of theinvention will become more apparent from the following more particulardescription of exemplary embodiments of the invention and theaccompanying drawings. The drawings are not necessarily to scale,emphasis instead being placed upon illustrating the principles of theinvention.

FIG. 1 is a schematic of the synthesis of secondary amine- anddiazeniumdiolate-functionalized PPI conjugates, wherein n represents thenumber of primary amines on the periphery of PPI dendrimers (n=8, 16,32, and 64).

FIGS. 2A and 2B are graphs that provide (2A) a real time NO releaseprofile for NO-releasing G4-PPI dendrimer conjugates; and (2B) a plot oft[NO] vs time for NO-releasing PPI dendrimer conjugates.

FIG. 3 is a schematic illustrating diazeniumdiolate-modifiedG5-PPI-PO/ACN (n=32) and it depicts the structurally different NO donorgroups and the surrounding nano environment of the “multi-donor” system(shown in a 1:1 ratio as an example).

FIG. 4 provides an 1H NMR spectra of G5-PPI-PO (c-64) (A), G5-PPI-ACN(a-64) (E), G5-PPI-PO/ACN at molar ratios of 7:3 (B), 5:5 (C), and 3:7(D). The actual compositions of PO and ACN incorporated into these threePPI conjugates are at molar ratios of 27:73 (B), 40:60 (C) and 60:40 (D)respectively, as determined by integrating two chemical shifts at 1.10and 2.80 ppm.

FIGS. 5A and 5B are graphs that provide (5A) an experimental plot ofpercent total NO released in PBS (pH=7.4) at 37° C. as a function oftime for G5-PPI-PO, G5-PPI-ACN and G5-PPI-PO/ACN conjugates; (5B) asimulated plot of percent total NO released for G5-PPI-PO/ACNconjugates.

FIG. 6 is a schematic illustrating a synthesis of a multi-donorco-condensed silica network according to one embodiment of theinvention.

FIG. 7 is a graph of controlled release for single donor co-condensedsilica and for various multi-donor combinations, with normalized totalNO released.

FIG. 8A is a graph of controlled release for single donor co-condensedsilica and for various multi-donor combinations, with normalized max NOconcentration.

FIG. 8B is a graph of controlled release for single donor co-condensedsilica and for various multi-donor combinations, with normalized max NOconcentration.

FIG. 9A is a graph that illustrates the difference in the releaseprofile of a Multi-donor Nitricil™ and a Nitricil™ mixture.

FIG. 9B is a graph that illustrations the difference in the degradationof a Multi-donor Nitricil™ and a Nitricil™ mixture in a pH 12 solution.

FIG. 10 is a schematic illustrating a multi-donor functionalizedchitosan macromolecule with a portion of the scaffold remainingacetylated.

DETAILED DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

The foregoing and other aspects of the present invention will now bedescribed in more detail with respect to the description andmethodologies provided herein. It should be appreciated that theinvention can be embodied in different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe embodiments of the invention and the appended claims, the singularforms “a”, “an” and “the” are intended to include the plural forms aswell, unless the context clearly indicates otherwise. Also, as usedherein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items. Furthermore,the term “about,” as used herein when referring to a measurable valuesuch as an amount of a compound, dose, time, temperature, and the like,is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1%of the specified amount. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, integers, steps, operations,elements, and/or components, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components, and/or groups thereof. Unless otherwise defined,all terms, including technical and scientific terms used in thedescription, have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. All patents,patent applications and publications referred to herein are incorporatedby reference in their entirety. In the event of conflicting terminology,the present specification is controlling.

The embodiments described in one aspect of the present invention are notlimited to the aspect described. The embodiments may also be applied toa different aspect of the invention as long as the embodiments do notprevent these aspects of the invention from operating for its intendedpurpose.

Chemical Definitions

As used herein the term “alkyl” refers to C₁₂₀ inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon chains, including for example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl,propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched”refers to an alkyl group in which a lower alkyl group, such as methyl,ethyl or propyl, is attached to a linear alkyl chain. Exemplary branchedalkyl groups include, but are not limited to, isopropyl, isobutyl,tert-butyl. “Lower alkyl” refers to an alkyl group having 1 to about 8carbon atoms (i.e., a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 toabout 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 carbon atoms. In certain embodiments, “alkyl” refers, in particular,to C₁₋₅ straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₅ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “aryl” is used herein to refer to an aromatic substituent thatcan be a single aromatic ring, or multiple aromatic rings that are fusedtogether, linked covalently, or linked to a common group, such as, butnot limited to, a methylene or ethylene functional group. The commonlinking group also can be a carbonyl, as in benzophenone, or oxygen, asin diphenylether, or nitrogen, as in diphenylamine. The term “aryl”specifically encompasses heterocyclic aromatic compounds. The aromaticring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether,diphenylamine and benzophenone, among others. In particular embodiments,the term “aryl” means a cyclic aromatic comprising about 5 to about 10carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a “substituted aryl”) withone or more aryl group substituents, which can be the same or different,wherein “aryl group substituent” includes alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl,aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio,alkylene, and —NR¹R″, wherein R¹ and R″ can each be independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term “substituted aryl” includes aryl groups,as defined herein, in which one or more atoms or functional groups ofthe aryl group are replaced with another atom or functional group,including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto. Specific examples of aryl groupsinclude, but are not limited to, cyclopentadienyl, phenyl, furan,thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole,isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine,quinoline, isoquinoline, indole, carbazole, and the like.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl,aryl, or substituted aryl, thus providing a heterocyclic group.Representative monocyclic cycloalkyl rings include cyclopentyl,cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings includeadamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl.

1 “Alkoxyl” refers to an alkyl-O— group wherein alkyl is as previouslydescribed. The term “alkoxyl” as used herein can refer to, for example,methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, f-butoxyl, andpentoxyl. The term “oxyalkyl” can be used interchangeably with“alkoxyl”. In some embodiments, the alkoxyl has 1, 2, 3, 4, or 5carbons.

“Aralkyl” refers to an aryl-alkyl group wherein aryl and alkyl are aspreviously described, and include substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Alkylene” refers to a straight or branched bivalent aliphatichydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; wherein each of q and r isindependently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R ishydrogen or lower alkyl; methylenedioxyl (—O—CH₂—O—); and ethylenedioxyl(—O—(CH₂)₂—O—). An alkylene group can have about 2 to about 3 carbonatoms and can further have 6-20 carbons.

“Arylene” refers to a bivalent aryl group. An exemplary arylene isphenylene, which can have ring carbon atoms available for bonding inortho, meta, or para positions with regard to each other, i.e.,respectively. The arylene group can also be napthylene. The arylenegroup can be optionally substituted (a “substituted arylene”) with oneor more “aryl group substituents” as defined herein, which can be thesame or different.

“Aralkylene” refers to a bivalent group that contains both alkyl andaryl groups. For example, aralkylene groups can have two alkyl groupsand an aryl group (i.e., -alkyl-aryl-alkyl-), one alkyl group and onearyl group (i.e., -alkyl-aryl-) or two aryl groups and one alkyl group(i.e., -aryl-alkyl-aryl-).

The term “amino” and “amine” refer to nitrogen-containing groups such asNR₃, NH₃, NHR₂, and NH₂R, wherein R can be alkyl, branched alkyl,cycloalkyl, aryl, alkylene, arylene, aralkylene. Thus, “amino” as usedherein can refer to a primary amine, a secondary amine, or a tertiaryamine. In some embodiments, one R of an amino group can be a cationstabilized diazeniumdiolate (i.e., NONO⁻X⁺).

The terms “cationic amine” and “quaternary amine” refer to an aminogroup having an additional (i.e., a fourth) group, for example ahydrogen or an alkyl group bonded to the nitrogen. Thus, cationic andquaternary amines carry a positive charge.

The term “alkylamine” refers to the -alkyl-NH₂ group.

The term “carbonyl” refers to the —(C═O)— group.

The term “carboxyl” refers to the —COOH group and the term “carboxylate”refers to an anion formed from a carboxyl group, i.e., —COO⁻.

The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups.

The term “hydroxyl” and “hydroxy” refer to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” or “thio” refers to the —SH group. The term “silyl”refers to a group comprising a silicon atom (Si).

As used herein the term “alkoxysilane” refers to a compound comprisingone, two, three, or four alkoxy groups bonded to a silicon atom. Forexample, tetraalkoxysilane refers to Si(OR)₄, wherein R is alkyl. Eachalkyl group can be the same or different. An “alkylsilane” refers to analkoxysilane wherein one or more of the alkoxy groups has been replacedwith an alkyl group. Thus, an alkylsilane comprises at least onealkyl-Si bond. The term “fluorinated silane” refers to an alkylsilanewherein one of the alkyl groups is substituted with one or more fluorineatoms. The term “cationic or anionic silane” refers to an alkylsilanewherein one of the alkyl groups is further substituted with an alkylsubstituent that has a positive (i.e., cationic) or a negative (i.e.anionic) charge, or can become charged (i.e., is ionizable) in aparticular environment (i.e., in vivo).

The term “silanol” refers to a Si—OH group.

The term “organic functional group” refers to any known organicfunctional groups, including all of the functional groups discussedabove, either alone or in combination. The organic functional group mayhave any suitable valency. In some cases, organic functional groups aredivalent, —R—, and in some cases, the organic functional groups aremonovalent, —R.

Multi-Donor Compounds

Provided according to some embodiments of the invention aremacromolecules that include at least two different nitric oxide (NO)donor structures of the same class. NO donors are of the same class ifthey have the same mechanism for release of nitric oxide (e.g.,N-diazeniumdiolate, S-nitrosothiol, etc.). NO donors of the same classmay be varied to provide different NO donor structures by altering thefunctional groups substituted on the NO donors. Accordingly, a nitricoxide donor structure as used herein includes at least one nitric oxidedonor and at least one functional group substituted on the nitric oxidedonor (“substituents”) that together provide the complete nitric oxidedonor structure and a signature NO-releasing behavior for that nitricoxide donor structure.

The functional group substituted on the NO donor can affect NO releasekinetics of the NO donor structure. The inventors have discovered thatcombining different NO donor structures on a macromolecular frameworkcan provide the ability to tune and/or vary the release rate of nitricoxide from the macromolecule based on the combination of the differentNO donor structures. In particular, in some embodiments, the combinationof different NO donor structures in a macromolecule can create a nano-or micro-environment that results in a distinct NO release profile,possibly as a result of an interaction of the effects of the differentsubstituent functional groups of the NO donor structures. Suchmacromolecules may also be referred to herein as “multi-donor”macromolecules. The hydrophobicity/hydrophilicity, the steric bulk andthe interaction of the different NO-donating structures with each otherand/or other functional groups on the macromolecule are some of thefactors which may affect the nitric oxide release rate. In some cases,the at least two different NO donor structures may be chosen to create adesired NO release profile for a particular use.

In some embodiments of the present invention, the combination ofdifferent NO donor structures of the same class on a macromolecule canresult in a release profile of nitric oxide that is the weighted sum ofthe release profiles of the individual NO donor structures. However, incertain embodiments, the release profile of the macromolecule differsfrom the weighted sum of the release profiles of the individual NO donorstructures. In such embodiments, release profiles that would nototherwise be achievable by combinations of NO releasing macromoleculeswith different NO donor structures may be realized.

In either of the above cases, the combination of different NO donorstructures on a macromolecule may increase thepredictability/probability that a given level of nitric oxide will bepresent at a given location over using a mix of macromolecules each witha single NO donor structures. For example, for a given unit volumecontaining a mixture of nitric oxide releasing small molecules havingdifferent release profiles, to achieve a desired release profile in agiven location within the volume, the small molecules at that locationmust not only be of sufficient quantity but must also be of the correcttypes and in the correct proportions to achieve the release profile.

For the case where the volume contains a mixture of nitric oxidereleasing macromolecules, the likelihood of achieving the local levelsof nitric oxide may be increased as each macromolecule releases morenitric oxide and, therefore, fewer macromolecules need be at the givenlocation to achieve the desired level of nitric oxide. Thus, even if theoverall release levels of nitric oxide are lower, the localized NOrelease may be higher, thus allowing for smaller doses to achievesimilar results. However, with a mixture of nitric oxide releasingmacromolecules, the problem of having the correct proportion to achievethe desired profile at a given location may still be present.

By combining different NO donor structures on a macromolecule, therelease profile for the macromolecule may be controlled such that anylocation within the volume will have the same or similar releaseprofiles. In such a case, the only requirement to achieve the desiredrelease profile at the given location is whether sufficientmacromolecules are at the location to achieve the desired levels.However, as discussed above, the macromolecule also increases thislikelihood at lower dose levels as each macromolecule would releasehigher levels of nitric oxide than would a small molecule NO donor.

In addition to providing performance benefits over mixtures of differentnitric oxide releasing donors, the multi-donor macromolecule may alsoprovide additional manufacturing benefits. For example, by creating amacromolecule with different NO donors, a single pharmaceuticalcomposition may be provided with, in some embodiments, thecharacteristics of the constituent NO donors and, in other embodiments,a new characteristic different from that of any of the constituents.Thus, issues of mixture uniformity, batch variability and the like maybe greatly reduced or eliminated.

In addition to allowing for the tuning and/or control of the releaseprofile of an NO releasing macromolecule, utilizing different NO donorstructures from the same class on a macromolecule may also improve thestability of the otherwise independent NO donor structure and/or thestability of the entire NO releasing macromolecule.

In some embodiments, the NO donor includes a diazeniumdiolate.Diazeniumdiolates react with proton donors to release NO atphysiological pH (˜7.4), and so the chemical functional groups that aresubstituted on the diazeniumdiolate donor may affect the ability of thediazeniumdiolate to react with the proton donors. Provided according tosome embodiments of the invention are macromolecules that include atleast two different diazeniumdiolate NO donor structures. In some cases,the at least two different diazeniumdiolate NO donor structures may bechosen to create a desired NO release profile for a particular use. Anysuitable diazeniumdiolate NO donor structures may be used, including forexample, C-based and N-based diazeniumdiolates, O₂-protecteddiazeniumdiolates, and the like.

In some embodiments, at least one of the diazeniumdiolate donorstructures includes the formula —R—N(NONO⁻X⁺)—R′, wherein R is adivalent organic functional group, R′ is a monovalent organic functionalgroup, and X⁺ is a monovalent cation. In some embodiments, R includesalkylene or arylalkylene, R′ includes alkyl, substituted alkyl,alkylnitrile, aryl, substituted aryl, alkylaryl, polyether and/oralkylamine, and X⁺ is includes Na⁺ or K⁺.

Examples of other NO donor classes include nitrosothiols, nitrosamines,hydroxyl nitrosamines, hydroxylamines, hydroxyureas, metal complexes,organic nitrites and organic nitrates.

As described above, embodiments of the invention provide an NO-releasingmacromolecule. As used herein, a “macromolecule” is defined as having amolecular weight of 500 daltons or greater. Any suitable size ofmacromolecule may be used. However, in some embodiments of theinvention, the hydrodynamic radius of the NO-releasing macromolecule iswithin a range from 0.01 nm to 1 nm, in some embodiments in a range from1 nm to 10 μm, in some embodiments in a range from 101 nm to 1000 nm,and in some embodiments, in a range from 1000 nm to 10 μm. In someembodiments, the hydrodynamic radius is greater than 10 μm, in someembodiments, in a range from 10 μm to 100 μm, in some embodiments,greater than 100 μm, and in some embodiments, greater than 1000 μm.

Dendrimers

In some embodiments of the invention, the NO-releasing macromoleculeincludes a dendrimer. Any suitable dendrimer may be used, including, forexample, polypropyleneimine (PPI) dendrimer; a polyamidoamine (PAMAM)dendrimer; polyarylether dendrimer; polypeptide dendrimer; polyamidedendrimer; dendritic polyglycerol; and triazine dendrimer.

Any suitable method may be used to synthesize the dendrimers. Particulardendrimers and methods for forming the same are described in detail inthe examples below. Other methods of synthesizing dendrimers are knownin the art, and may be used to form multi-donor dendrimers.

In some embodiments, the dendrimer includes at least two differentdiazeniumdiolate NO donor structures. In some cases, the dendrimer mayinclude a relatively fast NO releasing diazeniumdiolate in combinationwith a relatively slow diazeniumdiolate. Moderate/fast and moderate/slowcombinations may also be desirable in some cases. In particularembodiments, at least one of the diazeniumdiolate NO donor structureshas a NO release half life that falls within a range from 30 seconds to10 minutes and at least one diazeniumdiolate NO donor structure that hasa NO release half life of greater than 60 minutes (e.g., that fallswithin a range from 60 minutes to 4 days), in an aqueous solution at pH7.4 and 37° C. In some embodiments, at least one of the diazeniumdiolateNO donor structures has a NO release half life in a range from 30seconds and 10 minutes and at least one diazeniumdiolate NO donorstructure has a NO release half life greater than 10 minutes but lessthan or equal to 60 minutes, in an aqueous solution at pH 7.4 and 37° C.In some embodiments, at least one of the diazeniumdiolate NO donorstructures has a NO release half life of more than 10 minutes but lessthan or equal to 60 minutes and at least one diazeniumdiolate NO donorstructure has a NO release half life of greater than 60 minutes, in anaqueous solution at pH 7.4 and 37° C.

The maximum flux and NO release profile may also be varied in themulti-donor dendrimers, such that at least one of the diazeniumdiolateNO donor structures has a maximum flux of NO in a range from 2000 ppbNO/mg to 20,000 ppb NO/mg and a half life in a range from 0.1 to 1 hr,and at least one of the diazeniumdiolate NO donor structures has amaximum flux of NO in a range from 100 ppb NO/mg to 2000 ppb NO/mg and ahalf life in a range from 1 hr to 5 hr in an aqueous solution at pH 7.4and 37° C. The structure, size, and hydrophobicity of the dendrimer canbe varied to affect NO release.

As used herein, the values for the “NO release half life” and the“maximum flux” of a diazeniumdiolate NO donor structure are those of thecorresponding dendrimer that includes only that diazeniumdiolate. Itwill be understood that these properties will be shifted in amulti-donor dendrimer due to the contribution of the otherdiazeniumdiolate NO donor structures.

In particular embodiments of the invention, the dendrimer includes atleast one diazeniumdiolate NO donor structure having the structure:

wherein R is —CN, —COO(CH₂CH₂O)₆₋₁₂H, —CH₃, —CH₂CH₃, -Ph, —C₆H₁₂CH═CH₂;Z is —H or —OH; and X is Na⁺ or K⁺.

Co-Condensed Silica

In some embodiments of the invention, the NO-releasing macromoleculeincludes co-condensed silica. Any suitable co-condensed silica having atleast two different NO donor structures bound thereto may be used. Thematerials and methods that may be used to create NO-releasingco-condensed silica particles containing one nitric oxide donorstructure throughout are described in U.S. Patent ApplicationPublication No. 2009/0214618, the disclosure of which is incorporated byreference herein in its entirety. Examples of how to form multi-donorco-condensed silica are described below.

In some embodiments, each diazeniumdiolate NO donor structure may beformed from an aminoalkoxysilane by a pre-charging method, and theco-condensed siloxane network may be synthesized from the condensationof a silane mixture that includes an alkoxysilane and at least twodifferent NO-loaded aminoalkoxysilanes to form a multi-donorco-condensed siloxane network. As used herein, the “pre-charging method”means that the aminoalkoxysilane is “pretreated” or “precharged” withnitric oxide prior to the co-condensation with alkoxysilane. In someembodiments, pre-charging with nitric oxide may be accomplished bychemical methods. In some embodiments, the “pre-charging” method can beused to create co-condensed siloxane networks and materials more denselyfunctionalized with NO-donors.

The co-condensed siloxane network can be silica particles having auniform size, a collection of silica particles with a variety of size,amorphous silica, a fumed silica, a nanocrystalline silica, ceramicsilica, colloidal silica, a silica coating, a silica film, organicallymodified silica, mesoporous silica, silica gel, bioactive glass, or anysuitable form or state of silica.

In some embodiments, the alkoxysilane is a tetraalkoxysilane having theformula Si(OR)₄, wherein R is an alkyl group. The R groups can be thesame or different. In some embodiments the tetraalkoxysilane is selectedas tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS).In some embodiments, the aminoalkoxysilane has the formula:R″—(NH—R′)_(n)—Si(OR)₃, wherein R is alkyl, R′ is alkylene, branchedalkylene, or aralkylene, n is 1 or 2, and R″ is selected from the groupconsisting of alkyl, cycloalkyl, aryl, and alkylamine.

In some embodiments, the at least two different aminoalkoxysilanes areeach independently selected fromN-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3);N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3);(3-trimethoxysilylpropyl)di-ethylenetriamine (DET3);(aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3);[3-(methylamino)propyl]trimethoxysilane (MAP3);N-butylamino-propyltrimethoxysilane(n-BAP3);t-butylamino-propyltrimethoxysilane(t-BAP3);N-ethylaminoisobutyltrimethoxysilane (EAiB3);N-phenylamino-propyltrimethoxysilane (PAP3); andN-cyclohexylaminopropyltrimethoxysilane (cHAP3).

In some embodiments, at least one of the two different aminoalkoxysilanehas the formula: NH[R′—Si(OR)₃]₂, wherein R is alkyl and R′ is alkylene.In some embodiments, at least one of the aminoalkoxysilanes is selectedfrom bis(3-triethoxysilylpropyl)amine,bis-[3-(trimethoxysilyl)propyl]amine andbis-[(3-trimethoxysilyl)propyl]ethylenediamine.

In some embodiments, as described herein above, the at least twodifferent aminoalkoxysilanes are precharged, either together orseparately, for NO-release and the amino group is substituted by adiazeniumdiolate. Therefore, in some embodiments, the aminoalkoxysilanehas the formula: R″—N(NONO⁻X⁺)—R′—Si(OR)₃, wherein each R isindependently H, alkyl, silyl or aryl, R′ is a divalent organicfunctional group, R″ is a monovalent organic functional group, and X⁺ isa monovalent cation. In some embodiments, each R is independently methylor ethyl, R′ is alkylene or arylalkylene, R″ is alkyl, substitutedalkyl, alkylnitrile, aryl, substituted aryl, alkylaryl, polyether and/oralkylamine, and X⁺ is a cation selected from the group consisting ofNa⁺, K⁺, Cs⁺, Li⁺, NH₄+, or other quaternary ammonium cation.

In some embodiments of the invention, one or more of thediazeniumdiolate-functional aminoalkoxysilanes may be O²-protected priorto the preparation of the nitric oxide-releasing macromolecules. SuchO²-protected diazeniumdiolate functional aminoalkoxysilanes may have theformula: R″—N(NONO—R′″)—R′—Si(OR)₃, wherein each R is independently H,alkyl, silyl or aryl, R′ is a divalent organic functional group, R″ is amonovalent organic functional group, and R′″ is a protecting group thatimparts enzymatic, photolytic, or thiolation triggering mechanisms. Suchprotecting groups are known to those skilled in the art of formingO²-protected diazeniumdiolates.

The chemical composition of the siloxane network, (e.g., amount or thechemical composition of the aminoalkoxysilane), the porosity of thesilica network within the macromolecular structure, the size of theco-condensed silica particles, and the nitric oxide charging conditions(e.g., the solvent and base) can be varied to optimize the amount andduration of nitric oxide release. Thus, in some embodiments, thecomposition of the silica particles can be modified to regulate thehalf-life of NO release from silica particles with half-lives of nitricoxide release ranging from slow, defined by t_(1/2) values greater than60 minutes, to moderate, defined by t_(1/2) values greater than 10minutes and less than or equal to 60 minutes, to fast, defined byt_(1/2) values ranging from 30 seconds to 10 minutes. As describedabove, the combination of two or more different diazeniumdiolate NOdonor structures may alter the t_(1/2) values and the release profile.

In some embodiments, the multi-donor co-condensed silica may be presentas particles. In some embodiments, the particles have a size distributedaround a mean particle size of less than about 10 μm, and in someembodiments, have a particle size distributed around a mean particlesize of greater than about 10 μm, in some embodiments between 10 μm and100 μm, in some embodiments greater than 100 μm, and in some embodimentsgreater than 1000 μm.

In some embodiments of the invention, the multi-donor co-condensedsilica is also formed from at least one additional silane that modifiessurface charge and/or hydrophilicity/hydrophobicity of the co-condensedsilica product which affect the octanol/water partition coefficient ofthe macromolecular delivery vehicle. Any suitable alkoxysilane that mayimpart surface charge to the diazeniumdiolate-modified polysiloxanemacromolecule may be used. Thus, in some embodiments, the additionalalkoxysilane may include a cationic alkoxysilane such as(2-N-benyzlaminoethyl)-3-aminopropyl-trimethoxysilane, hydrocholoride;bis(methoxyethyl)-3-trimethoxysilylpropyl-ammonium chloride;N—N-didecyl-N-methyl-N-(3-trimethoxysilyl)ammonium chloride;N-trimethyoxysilylpropyl-N,N,N-trimethyl ammonium chloride;octadecylbis(triethoxysilylpropyl)-ammonium chloride; andoctadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride. In someembodiments, the additional alkoxysilane may include an anionicalkoxysilanes such as 3-trihydroxysilylpropylmethyl phosphonate, sodiumsalt and carboxyethylsilanetriol, sodium salt.

Any suitable alkoxysilane that may impart hydrophilic properties to themulti-donor co-condensed silica may be used. Alkoxysilanes containingrepeat poly(ethylene)oxy groups may be used to increase the wetabilityof the NO-releasing particles thereby helping to improvebiocompatibility upon topical application and also enhance the rate ofwater uptake into the co-condensed siloxane coating. Surfacehydrophilicity can thus be utilized to enhance the NO-release kineticsof the diazeniumdiolated aminoalkoxysilane derivatives. Therefore, insome embodiments, the multifunctional alkoxysilane may include ahydrophilic silane such as N-triethoxysilylpropyl)-O-polyethyleneoxideurethane; N-3-[amino(polypropylenoxy)]aminopropyltrimethoxysilane;bis-[3-(triethoxysilylpropoxy)-2-hydroxypropoxy]polyethylene oxide;bis(3-triethoxysilylpropyl)polyethylene oxide (25-30);[hydroxy(polyethyleneoxy)propyl]-triethoxysilane; and2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane.

Any suitable alkoxysilane that may impart hydrophobic properties to themulti-donor co-condensed silica may be used. Hydrophobic silanes areknown to those skilled in the art to increase lipophilicity of particlesurfaces. In some embodiments, the additional alkoxysilane may includelinear alkyl, branched and cyclic alkylalkoxysilanes having at leastthree carbon atoms, substituted and unsubstituted phenyl alkoxysilanes,and fluorinated alkoxysilanes. Exemplary fluoroalkoxysilanes may includeheptadecafluoro-1,1,2-2-tetrahydrodecyl)triethoxysilane (shown in FIG.2I), (3,3,3-trifluoropropyl)trimethoxysilane,(perfluoroalkyl)ethyltriethoxysilane, nonafluorohexyltrimethoxysilane,nonafluorohexyltriethoxysilane,(tridecafluoro-1,1,2,2-tetrahydrooctyl)triethoxysilane, and(tridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane.

The hydrophilicity of the multi-donor co-condensed silica can beassessed by the use of a water/octanol partition coefficient. SeeOctanol-Water Partition Coefficients: Fundamentals and PhysicalChemistry, Vol. 2 of Wiley Series in Solution Chemistry. Chichester:John Wiley & Sons Ltd. (1997), which is herein incorporated by referencein its entirety. For example, hydrophobicdiazeniumdiolate-functionalized polysiloxane macromolecules may have awater/octanol partition coefficient in a range from 0.1 to 7, andhydrophilic diazeniumdiolate-functionalized polysiloxane macromoleculesmay have a water/octanol partition coefficient in a range from −2 to 0.

Other Macromolecular Scaffolds

Any other suitable NO-releasing macromolecular scaffold may be used,such as, for example, metallic clusters, including those described inU.S. Patent Application Publication No. 2009/0214618, the disclosure ofwhich is incorporated by reference herein in its entirety. In otherembodiments biodegradable macromolecular scaffolds may be usedincluding, for example, chitosan, cellulose, and other polysaccharidematerials.

Pharmaceutically Acceptable Compositions

In some embodiments, at least one NO-releasing macromolecule is presentin a pharmaceutically acceptable composition. A pharmaceuticallyacceptable composition, as defined herein, refers to a composition thatis suitable for application/delivery to a subject, such as a human,without undue side effects such as toxicity or irritation to the skin.Undue side effects are those that render the composition unsuitable forapplication/delivery to a subject because the harm from the side effectsoutweighs the benefits of the composition. In some embodiments,pharmaceutically acceptable compositions include at least oneNO-releasing macromolecule; optionally, at least one additionaltherapeutic agent; and at least one pharmaceutically acceptableexcipient.

The NO-releasing macromolecules may be present in pharmaceuticallyacceptable compositions according to embodiments of the invention at anysuitable concentration, but in some embodiments, the NO-releasingmacromolecules are present in the compositions at a concentrationsufficient to achieve the therapeutic goal. For example, in some cases,the concentration is sufficient to reduce inflammation, promote healingand/or to kill bacteria. As another example, in some cases, theconcentration is sufficient to decrease, eliminate or prevent acneand/or decrease sebum production. In some embodiments, the concentrationof NO-releasing macromolecules ranges from 0.1% to 20% w/w in thecomposition.

As described above, in some embodiments, pharmaceutically acceptablecompositions include at least one additional therapeutic agent, such asthose that have antimicrobial, anti-inflammatory, pain-relieving,immunosuppressant, vasodilating properties.

Pharmaceutically acceptable compositions in which the presentmulti-donor NO releasing macromolecule may be utilized include those asdescribed in U.S. application Ser. No. 12/580,418, filed Oct. 16, 2009;U.S. application Ser. No. 12/860,657, filed on Aug. 20, 2010; U.S.application Ser. No. 12/860,457, filed Aug. 20, 2010; and U.S.Provisional Patent Application No. 61/504,628; filed Jul. 5, 2011, thecontents of each of which are hereby incorporated by reference in theirentirety.

Methods of Treatment Using Compositions of the Invention

Provided according to embodiments of the invention are methods oftreating a subject by administering to the subject a macromolecule or apharmaceutical composition according to an embodiment of the invention.NO has been shown to be beneficial for the treatment of many medicalconditions. As such, any suitable medical condition may be treated witha composition of the invention. In particular, the compositionsdescribed herein may be beneficial for the treatment of dermatologicalconditions. Examples include microbial infections, inflammation, wounds,scars, acne, and the like. Other conditions are discussed in U.S.Publication No. 2009/0214618, which contents are incorporated byreference in their entirety. Dermatological conditions are alsodiscussed in U.S. Provisional Patent Application No. 61/504,634; filedJul. 5, 2011, the contents of which are herein incorporated by referencein their entirety.

In some embodiments, the pharmaceutically acceptable compositionaccording to embodiments of the invention may be applied topically tothe skin of the subject. Any portion of the subject's skin may betreated. However, in some embodiments, the subject's face is treated bya method described herein. Furthermore, in some embodiments, thesubject's trunk is treated by a method described herein.

Additionally, in some embodiments, the pharmaceutically acceptablecomposition according to embodiments of the invention is applied inanother manner, such as systemic application. As used herein, systemicapplication refers to application/delivery of the pharmaceuticallyacceptable composition throughout the body. Furthermore, in someembodiments, the pharmaceutically acceptable composition may beapplied/delivered to the subject parenterally, orally, buccally,subcutaneously, via inhalation, intratracheally, surgically,transdermally, or by any other method known in the art for introductionof a medicament to the body.

Subjects suitable to be treated with a method embodiment of theinvention include, but are not limited to, avian and mammalian subjects.Mammals of the present invention include, but are not limited to,canines, felines, bovines, caprines, equines, ovines, porcines, rodents(e.g. rats and mice), lagomorphs, primates, humans, and the like, andmammals in utero. Any mammalian subject in need of being treatedaccording to the present invention is suitable. Human subjects of bothgenders and at any stage of development (i.e., neonate, infant,juvenile, adolescent, adult) can be treated according to the presentinvention.

Illustrative avians according to the present invention include chickens,ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) anddomesticated birds (e.g., parrots and canaries), and birds in ovo.

The invention can also be carried out on animal subjects, particularlymammalian subjects such as mice, rats, dogs, cats, livestock and horsesfor veterinary purposes, and for drug screening and drug developmentpurposes.

In some embodiments, methods of treating a dermatological condition byadministering to a subject a pharmaceutical composition according to anembodiment of the invention may be performed in combination with anothertherapeutic regimen and/or in combination with other medicaments, suchas those that have antimicrobial, anti-inflammatory, pain-relieving,immunosuppressant, vasodilating properties, and/or anti-acne properties.For example, in the treatment of acne, other anti-acne agents such asretenoids, may be used in conjunction (prior, concurrently or after)with the application of the pharmaceutical composition of the invention.As such, in some embodiments of the invention, a patient may be treatedwith a composition described herein in combination with an additionaltherapeutic agent when the additional therapeutic agent is not in thesame composition. For example, in some embodiments, an additionaltherapeutic agent may be administered (e.g., topically, systemically,parenterally, orally, buccally, subcutaneously, via inhalation,intratracheally, surgically, transdermally, etc.), either concurrentlyand/or sequentially with application of the pharmaceutically acceptablecomposition.

In some embodiments of the invention, a pharmaceutically acceptablecomposition may be administered to the skin via spray delivery. Anon-aqueous delivery propellant may be used for water sensitiveNO-releasing compounds such as diazeniumdiolate-modified compounds.Further, in some embodiments, particular components of the medicamentsmay be separated at some point prior to application of the medicament.For example, a water reactive NO-releasing compound may be storedseparately from an aqueous component or propellant until application(e.g., via spraying or applying a gel). In some embodiments, theNO-releasing compounds may be combined with an aqueous constituent priorto application or the NO-releasing compounds and an aqueous constituentmay be applied to the skin sequentially.

In some embodiments, a composition that includes nitrosothiol-modifiedcompounds may be kept at a low temperature (e.g, <0° C.) to minimizethermal decomposition and NO release. The cold composition may then beapplied to the skin, and the elevated temperature of the skin may allowfor the release of NO. In some embodiments, the nitrosothiol may bepresent in a medicament (e.g., a hydrophilic formulation which may limitNO diffusion) such that it is stable at room temperature due to cageeffects, and then releases NO upon application to the skin. Light mayalso be applied to a medicament that includes nitrosothiol modifiedcompounds. The application of light in fluxes may be applied to createfluxes of NO.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention will be further illustrated by the followingnon-limiting examples.

EXAMPLES Dendrimers: Materials and General Considerations

Ethylenediamine (EDA), acrylonitrile (ACN), propylene oxide (PO),styrene oxide (SO), methyl acrylate (MA), poly(ethylene glycol) methylether acrylate (average Mn=480) (PEG), and 1,2-epoxy-9-decene (ED) werepurchased from Aldrich Chemical Company (Milwaukee, Wis.).1,6-Hexanediamine (HDA) and sodium methoxide (5.4 M solution inmethanol) was purchased from Acros Organics (Geel, Belgium). MattheyCatalysts (London, UK). Common laboratory salts and solvents werepurchased from Fisher Scientific (Pittsburgh, Pa.). All other materialswere used as received without further purification unless otherwisenoted. 1H nuclear magnetic resonance (NMR) spectra were recorded onBruker (400 MHz) and Varian (600 MHz) spectrometers. Hydrogenationreactions used for the synthesis of PPI-NH2 (e.g., from G2 to G5) werecarried out in a stainless steel high-pressure reactor purchased fromParr Instrument Company (Moline, Ill.). Agitation was provided by aTeflon-coated magnetic stirring bar. Heating was provided using aheating fabric wrapped around the reactor, and temperature wascontrolled using a temperature controller via a thermal coupler. Nitricoxide release was measured using Sievers 280i Chemiluminesce NitricOxide Analyzer (Boulder, Colo.).

Example 1: Synthesis of [G-0.5]-PPI-CN to [G-4.5]-PPI-CN

For the synthesis of [G-0.5]-PPI-CN, ethylenediamine (EDA, 25.0 mL,0.374 mol) and deionized water (263 mL) were placed in a 1000 mLround-bottomed flask. Acrylonitrile (ACN, 140 mL) was added in portionsof 20 mL with stirring for 15 min. The resulting mixture was refluxedfor 2 h, and then cooled to room temperature overnight. ACN was removedin vacuo at 40° C. [G-0.5]-PPI-CN was crystallized from the mixture andisolated by vacuum filtration. The crude product was recrystallized fromTHF/methanol as a white powder.

Representative 1HNMR data was as follows: (300 MHz, CDCl3): δ (ppm) 2.55(8H, NCH2CH2CN), 2.77 (4H, —NCH2CH2N—), 2.96 (t, 8H, —NCH2CH2CN).Synthesis of higher generation PPI-CN (e.g., from [G-1.5] to [G-4.5])was not significantly different from the synthesis of PPI-[G-0.5]-CN asdescribed above, with the exception that PPI-CN (e.g., from [G-1.5] to[G-4.5]) were usually viscous liquids and their purification processeswould normally require the use of preparative-scale chromatography. As aresult, the synthesis of higher generation PPI-CN was conducted in amodified manner. Higher generation PPI-CN (10.0 g) (e.g., from [G-1.5]to [G-4.5]) was dissolved in deionized water (50 mL) and THF (100 mL)prior to adding ACN (50 mL). If phase separation of the resultingsolution was observed, an additional amount of THF was added. Thereaction mixture was stirred at room temperature for 3 d; a small amountwas then removed for analysis by 1H NMR spectroscopy to determine theextent of reaction. If the reaction was incomplete, an additional amountof ACN (25 mL) was added stirring continued for 2-3 additional days.

Example 2: Synthesis of [G-1]-PPI-NH2 to [G-5]-PPI-NH2

For the synthesis of [G-1]-PPINH2, sponge cobalt catalyst (5.0-6.0 g)was washed with 10% KOH solution for 10 min, three times with de-ionizedwater, and twice with methanol prior to use. [G-0.5]-PPI-CN (10.0 g) wasplaced in a glass reactor sleeve and dissolved in THF (70 mL) andmethanol (30 mL). To this solution, the sponge cobalt catalyst preparedas described above (5.0-6.0 g) was added using a pipet. The reactorsleeve was then placed in the hydrogenation chamber with properstirring. The reactor was purged with house nitrogen (60 PSI) fivetimes, and then with hydrogen (400 PSI) twice. The reactor was chargedwith hydrogen to a pressure of 800 PSI, and heated to 100° C. Thehydrogen gas pressure was maintained at 1000 PSI throughout thereaction. After 3 h, the reaction mixture was cooled to roomtemperature. Hydrogen was slowly removed, and the reaction chamber waspurged once with house nitrogen. The resulting reaction mixture was thenfiltered to remove the cobalt catalyst, and solvent was removed invacuo. The final product was dried under vacuum overnight to yield[G-1]-PPI-NH2 as a colorless liquid.

Representative 1H-NMR data was as follows: (300 MHz, CDCl3): δ (ppm)1.52 (q, 8H, NH2CH2CH2CH2N—), 2.40 (t, 8H, NH2CH2CH2CH2N—), 2.44 (s, 4H,—NCH2CH2N—), 2.64 (t, 8H, NH2CH2CH2CH2N—). The approach to synthesis ofhigher generation PPI-NH2 (e.g., from [G-2] to [G-5]) was similar tothat of [G-1]-PPI-NH2 with the exception that a mixture of EDA and THF(50:50, v/v) was used as a solvent. After reaction, the solvent wasremoved in vacuo using 1-butanol as an azeotropic agent due to its highboiling point. The resulting higher generation PPI-NH2 (e.g., from [G-2]to [G-5]) was dried under vacuum overnight to yield a light yellowliquid. Mass analysis by ESI MS was as, follows: 744.73, 1657.64,3483.45 and 7142.4 for G2-PPI, G3-PPI, G4-PPI and G5-PPI, respectively.

Example 3: Synthesis of Secondary Amine-Functionalized PPI Dendrimers

100 mg PPINH₂ (e.g., from G2 to G5) was dissolved in 2 ml methanol in a10 ml vial. One equivalent of acrylonitrile (ACN), poly(ethylene glycol)methyl ether acrylate (average Mn=480) (PEG), propylene oxide (PO),styrene oxide (SO), or 1,2-epoxy-9-decene (ED) (e.g., with respect tomolar amount of primary amine functionality) was then added to the 10 mLvial. The solution was stirred at room temperature for 4 days. Solventwas removed under reduced pressure. Dendrimers were dissolved in waterfollowed by dialysis against water and lyophilization.

Representative 1H NMR data of secondary amine-functionalized G5-PPIconjugate formed via the reactions of G5-PPI-NH₂ with ACN, PEG, PO, SO,and ED (referred to hereafter as G5-PPI-ACN a-64, G5-PPI-PEG b-64,G5-PPI-PO c-64, G5-PPI-SO d-64, G5-PPI-ED e-64) were as follows:G5-PPI-ACN a-64: 1H NMR (400 MHz, CD3OD, a): 2.87 (NHCH2CH2CN), 2.82(NHCH2CH2CN), 2.60 (NCH2CH2CH2NH), 2.40 (NCH2CH2CH2NH), 1.60(NCH2CH2CH2NH). 13C NMR (400 MHz, CD3OD, a): 117, 52.4, 51.8, 44.5,33.3, 26.1, 23.6, 16.9. G5-PPI-PEG b-64: 1H NMR (400 MHz, CD3OD, a):2.60 (NCH2CH2CH2NH), 2.40 (NCH2CH2CH2NH), 1.60 (NCH2CH2CH2NH), 3.40-3.70(OCH2CH2O), 2.80 (CH2NHCH2CHCOOPEG), 2.65 (CH2NHCH2CHCOOPEG), 2.42(CH2NHCH2CHCOOPEG). 13C NMR (400 MHz, CD3OD, 8): 172, 71.6, 70.85, 69.3,60.1, 57.0, 51.4, 43.9, 39.1, 22.9. G5-PPI-PO c-64: 1H NMR (400 MHz,CD3OD, 8): 3.70 (CH2CH(OH)CH3), 2.60-2.62 (CH2CH(OH)CH3, NCH2CH2CH2NH),2.40 (NCH2CH2CH2NH), 1.60 (NCH2CH2CH2NH), 1.00 (CH2CH(OH)CH3). 13C NMR(400 MHz, CD3OD, a): 66.9, 58.2, 53.7, 52.8, 41.2, 30.8, 27.6, 24.9,21.8. G5-PPI-SO d-64: 1H NMR (400 MHz, CD3OD, a): 7.50-7.20(CH2CH(OH)Ph), 3.70 (CH2CH(OH)Ph), 2.72 (CH2CH(OH)Ph), 2.60(NCH2CH2CH2NH), 2.40 (NCH2CH2CH2NH), 1.60 (NCH2CH2CH2NH). 13C NMR (400MHz, CD3OD, a): 140.6, 128.2, 127.5, 127.3; 125.8, 71.9, 57.1, 52.4,45.6, 39.8, 26.3, 23.6. G5-PPI-ED e-64: 1H NMR (400 MHz, CD3OD, a): 5.74(CH2CH═CH2), 4.88 (CH2CH═CH2), 3.58 (NHCH2CH(OH)CH2), 2.60(NCH2CH2CH2NH), 2.40 (NCH2CH2CH2NH), 1.98 (CH2CH═CH2), 1.60(NCH2CH2CH2NH), 1.2-1.4 ((CH2)5CH2CH═CH2). 13C NMR (400 MHz, CD3OD, 8):140.0, 116.1, 70.5, 56.5, 52.4, 47.2, 39.2, 33.9, 30.2, 25.6, 24.9.

Example 4: Synthesis of N-Diazeniumdiolate-Functionalized PPI Dendrimers

One equivalent of 5.4 M sodium methoxide solution in methanol (e.g.,with respect to the molar amount of primary amine functionalities inPPI-NH2 used to synthesize these secondary amine-functionalized PPI) wasadded to a vial containing G1 to G5 secondary amine-functionalized PPIdendrimers in methanol (2 mL). The resulting reaction solution wascharged with 10 atm of NO while stirring in a stainless steel reactor.Prior to charging with NO, the reactor was flushed three times withargon followed by a series of three longer charge/discharge cycles withargon (3×10 min) to remove oxygen from the stirring solutions. Thereactor was then filled with 10 atm of NO (purified over KOH pellets for30 min to remove trace NO degradation procedures described above withargon to remove unreacted NO from the reaction solution products) atambient temperature. After 3 days, the NO was expunged using the samecharge/discharge Example 5: Characterization of NO Storage and Release

Aliquots (˜10-25 μL) of N-diazeniumdiolate-functionalized PPI as asolution in methanol (e.g., ˜7-200 mM) were added to 30 mL phosphatebuffered saline (PBS) (10 mM, pH=7.4) at 37° C. to initiate/measure NOrelease. Chemiluminescence data for the NO-releasing dendrimers wererepresented as: i) total amount of NO release (t[NO], μmol NO/mg andμmol NO/μmol of secondary amine-functionalized dendrimers); ii) maximumflux of NO release ([NO]max, ppb/mg of secondary amine-functionalizeddendrimers); iii) half-life (t½) of NO release; and, iv) conversionefficiency defined as percentages of amine functionalities in PPI (e.g.,from G1 to G5) converted to N-diazeniumdiolate functionality (e.g.,total moles of NO release divided by twice the molar amount of primaryamine functionalities in PPI-NH2 used initially to synthesize secondaryamine-functionalized dendrimer conjugates).

Dendrimer Results and Discussion

Chemical reactions of primary amine functionalities with organiccompounds are considered to be the most straightforward approach forpreparing secondary amine-containing compounds. Both the ring-openingreactions of primary amine-containing compounds with epoxides andconjugate-addition reactions of primary amine functionalities withα,β-unsaturated double bonds represent viable approaches for designingsecondary amine-functionalized PPI conjugates. We thus targeted thesynthesis of secondary amine-functionalized dendrimers usingring-opening and conjugate-addition reactions of PPI-NH₂ with PO, SO,ED, ACN and PEG (See FIG. 1).

The epoxides, acrylates and acrylonitrile used for conjugation werebased on sterics, hydrophobicity and biocompatibility. Of note, it isalso possible to yield tertiary amine adducts. In addition, we carriedout a series of model kinetic studies using NMR spectroscopy forconjugate-addition reactions of first generation G1-PPI-NH₂ with ACN,1,6-hexanediamine (HDA) with MA, and ring-opening reactions of HDA withPO to determine the suitability of conjugate-addition and ring-openingreactions for the synthesis of secondary amine-functionalized PPIdendrimers. The results of these studies revealed large differences inthe rates of reactions of G-1-PPI-NH₂ with ACN, and HDA with PO. Therate constants of the first conjugate-addition or ring-opening reaction(k₁) were substantially larger than those of the second reactions (k₂)(data not shown), providing strong support for the use of such reactions(e.g., over a period of four days, in a dilute solution, and at oneequivalent of ACN, epoxides, or acrylates with respect to molar amountof PPI-NH₂) to yield secondary amine-functionalized products suitablefor subsequent NO release studies.

The approach described above is based on dendrimer functionalization attheir exterior to yield secondary amine-functionalized PPI dendrimers. Apractical advantage of this approach is that the synthesis ofstructurally diverse secondary amine-functionalized dendrimers may allowthe identification of key properties for NO storage and release. Indeed,reactions of secondary amine-functionalized PPI (e.g., from G2 to G5)with NO under basic conditions (e.g., sodium methoxide) yieldedN-diazeniumdiolate NO donor-functionalized dendrimers with diverse NOrelease characteristics.

Chemiluminescence was used to characterize the NO storage and releaseproperties (e.g., in PBS, pH=7.4, 37° C.) for theN-diazenuimdiolate-modified PPI dendrimers. Representative NO releaseprofiles for these dendrimers are shown in FIGS. 2A and 2B. The workupof specific NO release parameters (e.g., total NO release, maximum flux,half-life and conversion efficiency) are provided in Table 1. Ingeneral, the NO release results reveal high NO storage capabilities(e.g., 0.9-3.8 μmol NO/mg) and a broad range of release kinetics (e.g.,NO release half-life from 0.8-4.9 h). Further inspection of these datareveals that the conversion efficiencies (e.g., 10-34%) of thedendrimers varied substantially based on the chemical modification. Asshown in Table 1, G2 to G5-PPI-SO (d-n-NO) were characterized by lowerNO donor formation (e.g., ˜10-15%) versus the other PPI dendrimers(e.g., ˜14-40%). The lower conversion efficiencies for PPI-SO (d-n-NO)may be attributed to a more sterically-hindered environment around theNO donor precursors (i.e., secondary amines), resulting in lower NO andbase accessibility to the amines during the NO charging process.

TABLE 1 Nitric oxide release characteristics for PPI dendrimers in PBS(pH =7.4) at 37° C.. Con- t[NO] t[NO] [NO]_(max) [NO]_(max) versionDendrimer (μmol NO/mg)^(a) (μmol NO/μmol)^(b) (ppb/mg)^(c)(ppb/μmol)^(d) t_(1/2)(h) (%) a-8-NO G2-PPI-ACN-NO 3.57 4.18 1529 17884.81 26.1 a-16-NO G3-PPI-ACN-NO 3.33 8.35 1100 2758 4.84 26.1 a-32-NOG4-PPI-ACN-NO 2.45 12.7 1547 7977 4.82 19.9 a-64-NO G5-PP1-ACN-NO 1.6817.7 881 9282 4.88 13.8 b-8-NO G2-PPI-PEG-NO 1.11 5.10 3771 17291 0.6731.9 b-16-NO G3-PPI-PEG-NO 1.08 10.1 2309 21564 1.11 31.6 b-32-NOG4-PPI-PEG-NO 1.37 25.8 4088 77035 0.84 40.3 b-64-NO G5-PPI-PEG-NO 1.1744.3 1886 71403 1.22 34.6 c-8-NO G2-PPI-PO-NO 2.99 3.63 17130 20725 0.3022.7 c-16-NO G3-PP1-PO-NO 3.22 8.38 9617 24888 0.62 26.2 c-32-NOG4-PPI-PO-NO 3.27 17.5 7762 41481 0.78 27.3 c-64-NO G5-PPI-PO-NO 3.7841.1 6839 74250 1.06 32.1 d-8-NO G2-PPI-SO-NO 1.14 1.95 8495 14496 1.4712.2 d-16-NO G3-PPI-SO-NO 0.91 3.30 2363 8462 0.97 10.3 d-32-NOG4-PPI-SO-NO 1.18 8.70 1720 12609 1.43 13.6 d-64-NO G5-PPI-SO-NO 1.2518.5 3315 32843 1.62 14.5 e-8-NO G2-PPI-ED-NO 1.95 3.87 5648 11178 0.8124.2 e-16-NO G3-PPI-ED-NO 1.64 6.75 2733 11279 1.71 21.1 e-32-NOG4-PPI-ED-NO 1.51 12.8 2401 20217 1.34 19.9 e-64-NO G5-PPI-ED-NO 1.8631.6 3190 54262 1.88 24.7 ^(a)total amount of NO release (μmol) permilligram of secondary amine-functionalized PPI. ^(b)total amount of NOrelease (μmol) per micromole of secondary amine-functionalized PPI.^(c)maximum flux of NO release (ppb) per milligram of secondaryamine-functionalized PPI. ^(d)maximum flux of NO release (ppb) permicromole of secondary amine-functionalized PPI.

The exterior modification also influenced the NO release kinetics. Forexample, both PPI-PO (c-n-NO) and PPI-PEG (b-n-NO) released NO rapidly(Table 1 and FIGS. 2A-2B). Isopropyl and PEG groups are hydrophilic andfacilitate water salvation that can be favorable to diazeniumdiolate NOdonor degradation, so these groups could provide for rapid NO releasekinetics. The data also indicate that the NO release half-lives forG2-G5 PPI-SO (d-n-NO) and PPI-ED (e-n-NO) are slightly longer thanPPI-PO (c-n-NO) and PPI-PEG (b-n-NO). The longer NO release for PPI-SO(d-n-NO) and PPI-ED (e-n-NO) correlates well with the increasedhydrophobic structure at the exteriors of these dendrimers.

The ACN modification for PPI dendrimer (a-n-NO) exhibited large NOstorage (e.g., ˜1.7-3.6 μmol NO/mg) and conversion efficiency (e.g.,˜14-26%) (Table 1). Of note, past studies have indicated that thereaction of cyano-containing compounds with NO at high-pressures underbasic conditions may yield C-diazeniumdiolate-functionalized products.Both NO and nitrous oxide (N₂O) may be release from C-diazeniumdiolatesin aqueous environments at low pH. In this context, it may be possiblethat the high conversion efficiencies for PPI-ACN (a-n-NO) arise fromthe contribution of NO released from C-diazeniumdiolate-functionalizedproducts. A series of experiments were thus carried out to probe thenature of the NO release from PPI-ACN (a-n-NO) using G0.5-PPI, acyano-containing compound without the capacity to formN-diazeniumdiolate due to the absence of secondary amines. The NOrelease from G0.5-PPI was ˜4.5×10⁻³ μmol NO/mg, providing strong supportthat the high NO storage and conversion efficiency for the ACN-modifieddendrimers are indeed the result of N-diazeniumdiolatefunctionalization.

The PPI-ACN (a-n-NO) analogues were also characterized as having thelongest NO release half-lives (e.g., ˜5 h). Given the hydrophilic natureof the cyano functionality, the extended NO release is not easilyattributed to water uptake. For example, the long half-lives ofN-diazeniumdiolate-functionalized small molecule derivatives (e.g.,dipropylenetriamine or DPTA-NO) have previously been attributed todiazeniumdiolate stabilization by neighboring cationic ammoniumfunctionalities as depicted in molecules A and B below.

In this manner, the presence of neighboring cationic functionalities(e.g., protonated imidates) for PPI-ACN (a-n-NO) dendrimers may provideadditional stabilization to the diazeniumdiolate functionality (A).Since sodium methoxide was used as the base for the reaction of PPI-ACN(a-n) with NO (to yield diazeniumdiloate-functionalized products), themethoxide anion may also serve as a nucleophile to react with the cyanogroup in PPI-ACN (a-n) and yield imidate adducts.⁵³ The transfer ofproton from solvent (e.g., methanol) to the relatively basic nitrogenatom in the resulting imidates might thus lead to protonated-imidatefunctionality (B) that is similar to cationic ammonium functionality inDPTA-NO (A).

As shown in Table 1, both the NO storage and maximum NO flux perdendrimer molecule increase as a function of dendrimer size (i.e.,generation). For example, the NO payload from G5-PPI-PO-NO (c-64—NO) was41.1 μmol/μmol dendrimer, much greater than G2-PPI-PO-NO (c-8—NO) (e.g.,3.63 μmol/μmol dendrimer). A similar trend is observed for maximum NOflux. These results reveal the capability of larger NO-releasing PPIdendrimers to deliver significant concentrations of NO. As synthesized,the amphiphilic secondary amine-functionalized dendrimers (e.g., PPI-SO,PPI-ED) possess a hydrophilic PPI core and hydrophobic periphery ofaromatic rings or long alkyl chains. Different from PPI-ACN, PPI—PO andPPI-PEG, these amphiphilic dendrimers may have more packed exterior inthe charging solvent due to the poor compatibility of the aromatic ringsor long alkyl chains with polar solvent (e.g., methanol).

To evaluate the ability to create tunable NO release profiles betweenthe release kinetics observed for single PPI dendrimer modifications, wedesigned multi-functionalized N-diazeniumdiolate-functionalizeddendrimer conjugates using G5-PPI-NH₂ with defined ratios of PO and/orACN at the exterior. Specifically, G5-PPI-NH₂ was reacted with either POexclusively (e.g., c-64), ACN exclusively (e.g., a-64), or threedifferent mixtures comprised of PO and ACN at molar ratios of 3:7, 5:5,and 7:3, respectively; the PO, ACN, or defined mixtures of PO and ACNwere one equivalent with respect to molar amount of primary aminefunctionalities in G5-PPI-NH₂. FIG. 3 illustrates that the two donorstructures may interact to form a unique NO-releasing nano-environment.

A series of ¹H NMR experiments were carried out on the products of thesereactions to determine the actual compositions of PO and ACN conjugatedto the G5-PPI—NH₂ (FIG. 4). A distinct resonance at 1.10 ppm wasapparent corresponding to the methyl protons in the isopropyl group ofthe products. The chemical shift of this peak was noted in the productsfor reactions with PO exclusively (e.g., c-64) and those with differentmixtures of PO and ACN. Further inspection of these data indicates thepresence of a second distinct peak at 2.80 ppm, formed upon G5-PPI-NH₂dendrimer reaction with either ACN exclusively (e.g., a-64) or definedmixtures of PO and ACN; this peak corresponds to the methylene protonsone carbon away from the cyano group of the products. The compositionsof PO and ACN incorporated in the products were 27/73 (c/a (27/73)),40/60 (c/a (40/60)), and 60/40 (c/a (60/40)), and indicate that the ACNwas incorporated at ratios greater than that in the reaction mixtureslikely due to more rapid reaction of G5-PPI-NH₂ with ACN versus PO.

The NO release from the G5-PPI-PO/ACN conjugates at PO/ACN molar ratiosof 27/73, 40/60, and 60/40, respectively, were intermediate to thosesynthesized based upon G5-PPI-NH₂ reactions with either PO (e.g., c-64)or ACN (e.g., a-64) alone. Furthermore, the NO release profiles wereinfluenced by the molar ratio of PO/ACN composition. For instance, theNO release was prolonged for the PPI conjugates modified with lowermolar ratio of PO/ACN. (2.90, 1.57, 1.10 h for 27/73, 40/60 and 60/40,respectively) (FIGS. 5A and 5B).

Simulated NO release profiles (expressed as percentages of total NOrelease, y_(a:b)) were determined by averaging the NO release profilesfor G5-PPI-PO (a-64) (y_(PO)) and G5-PPI-ACN (a-64) (y_(ACN)) weightedwith respect to the actual compositions of PO and ACN in these three PPIconjugates (expressed as molar ratios, a:b) (Eq. 1).

$\begin{matrix}{y_{a:b} = {{y_{PO} \times \frac{a}{a + b}} + {y_{ACN} \times \frac{b}{a + b}}}} & (1)\end{matrix}$

As shown in FIGS. 5A and 5B, the hybrid PPI conjugates werecharacterized by more rapid NO release than expected by the simulateddata (NO release half-lives of 3.13, 2.42, 1.65 for c/a (27/73), c/a(40/60) and c/a (60/40), respectively). These results demonstrate thatfunctionalization of PPI-NH₂ with a defined mixture of PO and ACN allowsfor further NO release tunability with access to NO release kineticsthat are intermediate to those formed upon reactions with either PO orACN exclusively.

The synthesis of diverse NO-releasing PPI dendrimers was achieved bychemical modification of exterior primary amines with ACN and PEG viaconjugation addition or PO, ED, SO via ring opening reactions. The NOrelease from these dendrimers demonstrated that size (i.e., generationnumber or molecular weight) and exterior structures (e.g., stericenvironment, hydrophobicity, etc.) play important roles in NO releasekinetics. Furthermore, the use of select NO donors with unique NOrelease kinetics and overall payloads may be exploited by utilizingthese synthetic modification to create “multi-donor” dendrimers. Thestructurally diverse dendritic scaffolds extend the range and scope ofsecondary amine functionalities that may be designed into PPIconjugates.

Co-Condensed Silica Examples Example 6

An example of a synthesis of multi-donor co-condensed silica will now beprovided and is shown schematically in FIG. 6. MAP3 diazeniumdiolate wasmixed with AEAP diazeniumdiolate. The former molecule is known for itsquick NO release and is able to rapidly establish and expose bacteria toa high concentration of nitric oxide, but the release lasts only a fewminutes. On the other hand, AEAP diazeniumdiolate has a rather slowrelease profile with a half life in PH=7.4 buffer at over 3.5 hours. Theconstant exposure of bacteria to NO can effectively inhibit growth,however, it does not offer a high initial nitric oxide concentrationthat may be toxic to existing bacteria. By combining the two silanesonto the same polymeric structure, a programmable release profile can beachieved for improved effect in, for example, an antimicrobialapplication.

A variety of single and multi-donor co-condensed silica macromoleculeswere synthesized. FIG. 7 shows the total NO released over a certain timeperiod from different NO-releasing co-condensed silica macromolecules,with normalized total loading. FIGS. 8A and 8B illustrate the samerelease profiles with normalized maximum NO concentration. FIG. 8Billustrates the same release profiles, with normalized maximum NOconcentration, as FIG. 8A, but is an expanded view of the releaseprofiles. All release profiles were collected with a Nitric OxideAnalyzer under physiological condition at pH=7.4 and 37° C.

In addition, physical mixtures of separately synthesized single donorco-condensed silica macromolecules were also prepared. The idea ofincorporating different NO-loaded aminosilanes onto one backbone hasseveral advantages over a physical mixture of two different co-condensedsilicas. One such advantage is homogeneity, and another is enhancedstability. FIGS. 9A and 9B illustrate the releasing curve of themulti-donor silica macromolecules and a physical mixture of the samedonors on separate co-condensed silica scaffolds. It is clear that inthe first hour shown the kinetics are different.

The co-condensed siloxane network with diazeniumdiolates incorporatedthroughout the macromolecule also has increased stability compared toeach individual particles prepared with only one NO-loaded. FIG. 9Bshows the degradation of the multi-donor silica slowed downsignificantly compare to the physical mixtures. This discovery may beextremely useful in increasing the ability to preserve the NO untiltriggered release at the location of targeted medical application. Whilenot wishing to be bound to any particular theory, the mechanism for thestability increase appears to be a coordinated effort of both sterichindrance of the proton attack and intramolecular hydrogen bondingbetween different silanes. The intramolecular protective interaction isnot available in the mixed solid, which resulted in a fasterdegradation.

To make aminosilane diazeniumdiolate monomers, the aminosilane is addedinto a high pressure reaction vessel together with 1 equivalent of NaOMe(25-30% w.w) MeOH solution. An equal volume of dried methanol is alsoadded. The reactor is pump/purged with argon at 100 psi for three timesbefore NO was pumped into the reactor. The initial reaction pressure isat least 300 psi and as high as 420 psi. The pressure is kept at thisrange with refill. The reaction is stopped at two hours and the contentis used in the following condensation reactions without furtherisolation.

To make NO-releasing co-condensed silica with programmed releaseprofile, the different silane diazeniumdiolate monomer solutions weremixed at the pre-set ratios. The mixture is then added into a flask withpre-cooled ethanol with nitrogen protection. The mixture is furthercooled to −15° C. and pre-set amount of backbone silane (TEOS or TMOS)is added. When the mixture temperature equilibriums to −15° C., theammonium hydroxide aqueous solution is added. The reaction is thenwarmed rapidly to 150 and stirred for additional 3 hours. The content isthen filtered. The solid is washed with dry ethanol twice and then driedextensively.

TABLE 2 An Example of Programmable Release from Two Silanes Sample HalfLife/ Total NO Composition Ratio minutes umol/mg Reference 1 MAP3/AEAP3100/0  4 4.5 10-7-132 2 MAP3/AEAP3 50/50 23 4.1 11-4-61 3 MAP3/AEAP333/66 70 3 11-4-59 4 MAP3/AEAP3 25/75 112 2.7 11-4-60 5 MAP3/AEAP3 0/100 210 5.1 11-4-29

It is clear that the diazeniumdiolate degradation is the same mechanismas the NO release path through proton initiated reaction illustratedbelow. Therefore, water/moisture are among the top reasons fordegradation. Experimental data have shown that somediazeniumdiolate-functionalized co-condensed silica are hygroscopic, andthe intrinsic water may be tightly bounded to the silicate in the backbone of the polymer. The hygroscopic property of thediazeniumdiolate-functionalized co-condensed silica may lead todegradation of the diazeniumdiolate.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

That which is claimed:
 1. A nitric oxide-releasing macromoleculecomprising at least two different NO donor structures of the same class.2. The nitric oxide-releasing macromolecule of claim 1, wherein the atleast two different NO donor structures have different nitric oxiderelease kinetics.
 3. The nitric oxide-releasing macromolecule of claim1, wherein the class is diazeniumdiolate donors or nitrosothiol donors.4. The nitric oxide-releasing macromolecule of claim 1, wherein thecompound comprises at least two different diazeniumdiolate donorstructures.
 5. The nitric oxide-releasing macromolecule of claim 4,wherein at least one of the diazeniumdiolate donor structures has theformula —R—N(NONO⁻X⁺)—R′, wherein R is a divalent organic functionalgroup, R′ is a monovalent organic functional group, and X⁺ is amonovalent cation.
 6. The nitric oxide-releasing macromolecule of claim5, wherein R is selected from alkylene or arylalkylene; R′ is selectedfrom alkyl, substituted alkyl, alkylnitrile, aryl, substituted alkyl,alkylaryl, polyether and alkylamine; and X⁺ is selected from Na⁺ and K⁺.7. The nitric oxide-releasing macromolecule of claim 4, wherein at leastone diazeniumdiolate donor structure comprises the structure:

wherein R is —CN, —COO(CH₂CH₂O)₆₋₁₂H, —CH₃, —CH₂CH₃, -Ph, —C₆H₁₂CH═CH₂;Z is —H or —OH; and X is Na⁺ or K⁺.
 8. The nitric oxide-releasingmacromolecule of claim 1, wherein the nitric oxide-releasing compoundcomprises a dendrimer.
 9. The nitric oxide-releasing macromolecule ofclaim 8, wherein the dendrimer comprises at least one of apolypropyleneimine dendrimer; a polyamidoamine (PAMAM) dendrimer; apolyarylether dendrimer; a polypeptide dendrimer; a polyamide dendrimer;a dendritic polyglycerol; and a triazine dendrimer.
 10. The nitricoxide-releasing macromolecule of claim 9, wherein the compound comprisesat least two different diazeniumdiolate donor structures.
 11. The nitricoxide-releasing macromolecule of claim 10, wherein at least one of thediazeniumdiolate donor structures has a NO release half life in a rangefrom 30 seconds to 10 minutes and at least one diazeniumdiolate donorstructure has a NO release half life of greater than 60 minutes, asdetermined in aqueous buffer at pH 7.4 and 37° C.
 12. The nitricoxide-releasing macromolecule of claim 10, wherein at least one of thediazeniumdiolate donor structures has a NO release half life in a rangefrom 30 seconds to 10 minutes and at least one diazeniumdiolate donorstructure has a NO release half life greater than 10 minutes but less orequal to 60 minutes, as determined in aqueous buffer at pH 7.4 and 37°C.
 13. The nitric oxide-releasing macromolecule of claim 10, wherein atleast one of the diazeniumdiolate donor structures has a NO release halflife of more than 10 minutes but less or equal to 60 minutes and atleast one diazeniumdiolate donor structure has a NO release half lifegreater than 60 minutes, as determined in aqueous buffer at pH 7.4 and37° C.
 14. The nitric oxide-releasing macromolecule of claim 10, whereinat least one of the diazeniumdiolate donor structures has a maximum fluxof NO in a range from 2000 ppb NO/mg to 20,000 ppb NO/mg and a half lifein a range from 0.1 to 1 hour, and at least one diazeniumdiolate donorstructure has a maximum flux of NO in a range from 100 ppb NO/mg to 2000pp NO/mg and a half life in a range from 1 hour to 5 hours, asdetermined in aqueous buffer at pH 7.4 and 37° C.
 15. The nitricoxide-releasing macromolecule of claim 11, wherein at least one of thediazeniumdiolate donor structures comprises the structure:

wherein R is —CN, —COO(CH₂CH₂O)₆₋₁₂H, —CH₃, —CH₂CH₃, -Ph, —C₆H₁₂CH═CH₂;Z is —H or —OH; and X is Na⁺ or K⁺.
 16. The nitric oxide-releasingmacromolecule of claim 1, wherein the nitric oxide-releasing compoundcomprises co-condensed silica.
 17. The nitric oxide-releasingmacromolecule of claim 16, wherein the compound comprises at least twodifferent diazeniumdiolate donor structures.
 18. The nitricoxide-releasing macromolecule of claim 17, wherein at least one of thediazeniumdiolate donor structures has a NO release half life in a rangefrom 30 seconds to 10 minutes and at least one diazeniumdiolate donorstructure has a NO release half life of greater than 60 minutes, asdetermined in aqueous buffer at pH 7.4 and 37° C.
 19. The nitricoxide-releasing macromolecule of claim 17, wherein at least one of thediazeniumdiolate donor structures has a NO release half life in a rangefrom 30 seconds to 10 minutes and at least one diazeniumdiolate donorstructure has a NO release half life greater than 10 minutes but less orequal to 60 minutes, as determined in aqueous buffer at pH 7.4 and 37°C.
 20. The nitric oxide-releasing macromolecule of claim 17, wherein atleast one of the diazeniumdiolate donor structures has a NO release halflife of more than 10 minutes but less or equal to 60 minutes and atleast one diazeniumdiolate donor structure has a NO release half lifegreater than 60 minutes, as determined in aqueous buffer at pH 7.4 and37° C.
 21. The nitric oxide-releasing macromolecule of claim 17, whereinat least one of the diazeniumdiolate donor structures has a maximum fluxof NO in a range from 2000 ppb NO/mg to 20,000 ppb NO/mg and a half lifein a range from 0.1 to 1 hour, and at least one diazeniumdiolate donorstructure has a maximum flux of NO in a range from 100 ppb NO/mg to 2000pp NO/mg and a half life in a range from 1 hour to 5 hours, asdetermined in aqueous buffer at pH 7.4 and 37° C.
 22. The nitricoxide-releasing macromolecule of claim 17, wherein at least onediazeniumdiolate donor structure comprises

wherein n is 1-5 and X is Na⁺ or K⁺.
 23. The nitric oxide-releasingmacromolecule of claim 17, wherein the silica is present as particleshaving a mean particle size of less than 10 μm.
 24. A pharmaceuticalcomposition comprising the nitric oxide-releasing macromolecule of claim1, at least one excipient and, optionally, at least one additionaltherapeutic agent.
 25. A kit comprising at least two pharmaceuticalcompositions of claim
 24. 26. A wound dressing comprising a polymermatrix and the NO releasing macromolecule of claim
 1. 27. A kitcomprising at least two wound dressings of claim
 26. 28. A method oftreating a dermatological condition, comprising topically administeringthe NO releasing macromolecule of claim 1 in an amount effective toactively promote healing of a wound associated with the dermatologicalcondition.
 29. The method of claim 28, wherein the dermatologicalcondition comprises at least one of a wound, microbial infection andinflammation.
 30. The method of claim 28, wherein the dermatologicalcondition comprises acne.